The inventive apparatus and method described herein utilizes the concept of highly selective biochemical reactions between two types of complementary biological cells leading to the formation of large clumps or agglutinates and a separation concept based on different dimensions of the clumps and the biological cells and particulates not involved in the reaction. Antigens and corresponding specific antibodies, for example, are two such types of complementary biological cells, which when reacted, lead to the formation of large agglutinates.
In general, the antigen-antibody reaction occurs in two stages.sup.(1). In the first stage, an individual antigen combines with a complementary antibody. If specific antibodies are selected for a particular type of antigen to be removed or separated from a fluid, such as blood, the first-stage reaction may take place in a fraction of a second. Furthermore, the antibodies will react specifically with antigen cells which are complementary to each other and not with other cells of different species. Numerous papers have been published to demonstrate the extremely selective affinity between specific antibody and antigen cells. This selectivity in reaction is utilized in this invention.
The second stage antigen-antibody reaction involves the formation of large agglutinates or clumps, containing a large and often interconnected chain of antigen-antibody molecules. As early as in 1934, Marrack.sup.(2) described the reaction between antigen and antibody cells resulting in precipitation in terms of the building up of aggregates of large sizes. Marrack pointed out that if the antibody has more than one valency, it will be possible for antigens and antibodies to be bound together in the form of a coarse lattice.
The study in the field of second state antigen-antibody reaction leading to precipitation or agglutination was also made long ago by Heidelberger.sup.(3) who originally made use of purified capsular polysaccharide of the type III pneumococcus as antigens. The reactions were observed when an increasing amount of antigens were added to a constant amount of antiserum or antibody. After a time, sufficient for the complete reaction to occur, the precipitates were washed free of uncombined reactant and the total antigen content was estimated. It was seen that the amount of antibody precipitate increased to a maximum with the increasing addition of antigen and then suddenly this amount declined so that in the extreme antigen-excess case, no precipitate was formed. For a given antigen and the corresponding complementary antibody, then, there appears to be an optimal proportion of antigen to antibody cells which yield the largest agglutinates.
The second state antigen-antibody reaction is utilized in this invention so that physically large agglutinates or clumps can be obtained. The formation of large clumps of antigen and antibody molecules is believed to result from multiple reactions which can be described as follows:
______________________________________ A + S -- AS AS + A -- ASA ASA + A -- ASA A ASA ASA + S -- AS AS ______________________________________
where A and S denote respectively the antigen and antiserum (antibody) molecules. Indeed other reactions, besides those shown above, are possible.
The formation of large clumps of antigen and antibody has been observed by a large number of researchers. From such observations it appears that the dimensions of the antigen-antibody clump and the rate of clump formation depend on several factors such as antigen-antibody cells ratio, electrolyte concentration of the solution, temperature, antibody valency, etc. A polarizing electromagnetic field.sup.(4) and stirring can also affect clump sizes and their formation rates.
The concentration of antigens and antibodies in the solution, perhaps, affect the clump formation mast markedly. It is well known, for example, that the precipitation or agglutination varies in composition according to the proportions of antigen and antibody in the reacting mixture. If antibody is present in excess, the precipitation will contain relatively more of this component and vice versa. It is seen that if the valency of the antigen is N and that of the antibody is 2, there will be increasing chances of building large aggregates up to a point where S/A ratio is about N/2. It may be recognized that this combination of two substances to form a compound of variable composition according to the proportions in which they are mixed has no counterpart in chemical reactions of small molecules, but it is more akin to the polymerization of plastics. It is, of course, essential that the antigen be multivalent and the antibody be at least bivalent for the lattice hypothesis to work in this way.
An application of time-varying electromagnetic field shows possibilities of enhancing the rate of agglutination process. For example, large aggregates of non-spherical dielectric particles including biological cells have been made by the application of a time-varying electromagnetic field. This process is often referred to as pearl-chain formation. The formation of large aggregates of antigen and antibody molecules could be obtained by similar techniques. The pearl-chain formation is regarded as a direct consequence of increasing the free energy of the biological cells in solution by a non-thermal energy source such as the electromagnetic field. Since the energy that can be imparted by an electromagnetic field to the biological cells depends on the coupling mechanism, which in turn depends on the frequency and polarization of the electromagnetic field for a given set of antigens and antibodies, the average time required to form a clump of a given dimension can be considerably reduced if an appropriate electromagnetic field is impressed on the solution.
In addition, adaptation of complement fixation procedures, provisions of stirring and a reduction of hapten concentration in the antibody solution that normally terminates the antigen-antibody chain could be used to increase the agglutination and reduce the time required for the second-stage antigen-antibody reaction.
Besides antibodies, agglutinins have been developed such that clumps can be formed following a highly specific reaction of such agglutinins with certain complementary biological cells in a solution. Burger.sup.(5) for example, reported an agglutinin prepared from wheat germ lipase which reacted with tissue culture cell lines that were transformed by a tumor virus, while under identical conditions, their untransformed parent cell lines did not agglutinate. These and similar agglutinins can be used to form clumps and to separate specific biological cells.
Once large clumps containing the biological cells to be removed or separated are formed, a filter, that discriminates the filtrate against the residue based on their grossly different sizes, can be used for the final separation of the cells. Various mechanizations of the selective cell separation or cell removal in a closed-loop fluid flow path, containing the source of fluid, a reaction chamber where clumps of the cells to be removed are formed through a highly specific biochemical reaction, and a filter chamber where the clumps are retained permitting the fluid along with non-reacted cells and particulates to return to the source, are embodied in the invention.